Semisynthesis of Functional Glycosylphosphatidylinositol-Anchored Proteins

Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a conserved phosphoglycan that is modified in a cell- and tissue-specific manner. GPI complexity suggests roles in biological processes and effects on the attached protein, but the difficulties to get homogeneous material have hindered studies. We disclose a one-pot intein-mediated ligation (OPL) to obtain GPI-anchored proteins. The strategy enables the glypiation of folded and denatured proteins with a natural linkage to the glycolipid. Using the strategy, glypiated eGFP, Thy1, and the Plasmodium berghei protein MSP1₁₉ were prepared. Glypiation did not alter the structure of eGFP and MSP1₁₉ proteins in solution, but it induced a strong pro-inflammatory response in vitro. The strategy provides access to glypiated proteins to elucidate the activity of this modification and for use as vaccine candidates against parasitic infections.     

Synthetic Strategies for Artificial Lipidation of Functional Proteins

Biosynthesis of natural lipidated proteins is linked to important signal pathways, and therefore analyzing protein lipidation is crucial for understanding cellular functions. Artificial lipidation of proteins has attracted attention in recent decades as it allows modulation of the amphiphilic nature of the protein of interest, and is used in the design of drug-delivery systems containing antibodies anchored on a lipid bilayer carrier. However, the intrinsic hydrophobicity of lipids makes the synthesis of lipid–protein conjugates challenging with respect to the yield and selectivity of the lipidation. In this Minireview, the development of chemical and enzymatic synthetic strategies for the preparation of a range of lipid–protein conjugates that do not compromise the functions of the proteins are discussed as well as applications of the conjugates.     

Semisynthesis of Functional Glycosylphosphatidylinositol‐Anchored Proteins

Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a conserved phosphoglycan that is modified in a cell‐ and tissue‐specific manner. GPI complexity suggests roles in biological processes and effects on the attached protein, but the difficulties to get homogeneous material have hindered studies. We disclose a one‐pot intein‐mediated ligation (OPL) to obtain GPI‐anchored proteins. The strategy enables the glypiation of folded and denatured proteins with a natural linkage to the glycolipid. Using the strategy, glypiated eGFP, Thy1, and the Plasmodium berghei protein MSP1₁₉ were prepared. Glypiation did not alter the structure of eGFP and MSP1₁₉ proteins in solution, but it induced a strong pro‐inflammatory response in vitro. The strategy provides access to glypiated proteins to elucidate the activity of this modification and for use as vaccine candidates against parasitic infections.     

Pushing the Boundaries of Chemical Protein Synthesis: The Case of Ubiquitin Chains and Polyubiquitinated Peptides and Proteins

Chemical synthesis offers unique opportunities to prepare proteins with precise control of the atomic composition. Thanks to recent breakthroughs in synthetic methods, the preparation of large and complex proteins composed of 200–300 residues has now become possible. With these advances, a unique toolbox has been created to enable chemical biologists to investigate proteins that are difficult or even impossible to achieve otherwise, such as posttranslationally modified proteins and proteins composed of d ‐amino acids. In this review we describe the latest achievements in constructing protein conjugates of record sizes, such as those that are involved in the ubiquitin system.

     

Polyphenol-Mediated Assembly of Proteins for Engineering Functional Materials

Functional materials composed of proteins have attracted much interest owing to the inherent and diverse functionality of proteins. However, establishing general techniques for assembling proteins into nanomaterials is challenging owing to the complex physicochemical nature and potential denaturation of proteins. Here, a simple, versatile strategy is introduced to fabricate functional protein assemblies through the interfacial assembly of proteins and polyphenols (e.g., tannic acid) on various substrates (organic, inorganic, and biological). The dominant interactions (hydrogen-bonding, hydrophobic, and ionic) between the proteins and tannic acid were elucidated; most proteins undergo multiple noncovalent stabilizing interactions with polyphenols, which can be used to engineer responsiveness into the assemblies. The proteins retain their structure and function within the assemblies, thereby enabling their use in various applications (e.g., catalysis, fluorescence imaging, and cell targeting).     

Nonenzymatic recombination of RNA by means of transesterification

Coupled nonenzymatic cleavage/ligation of oligoribonucleotides catalyzed by magnesium ions afforded longer RNA molecules with a new sequence. The efficiency of formation of ligation products reaches 6%. The possible role of this reaction in the evolution of the RNA world is discussed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2413–2419, December, 2007.     

Polyphenol‐Mediated Assembly of Proteins for Engineering Functional Materials

Functional materials composed of proteins have attracted much interest owing to the inherent and diverse functionality of proteins. However, establishing general techniques for assembling proteins into nanomaterials is challenging owing to the complex physicochemical nature and potential denaturation of proteins. Here, a simple, versatile strategy is introduced to fabricate functional protein assemblies through the interfacial assembly of proteins and polyphenols (e.g., tannic acid) on various substrates (organic, inorganic, and biological). The dominant interactions (hydrogen‐bonding, hydrophobic, and ionic) between the proteins and tannic acid were elucidated; most proteins undergo multiple noncovalent stabilizing interactions with polyphenols, which can be used to engineer responsiveness into the assemblies. The proteins retain their structure and function within the assemblies, thereby enabling their use in various applications (e.g., catalysis, fluorescence imaging, and cell targeting).     

Chemical Synthesis of Phosphorylated Ubiquitin and Diubiquitin Exposes Positional Sensitivities of E1‐E2 Enzymes and Deubiquitinases

Modification of ubiquitin by phosphorylation extends the signaling possibilities of this dynamic signal, as it could affect the activity of ligases and the processing of ubiquitin chains by deubiquitinases. The first chemical synthesis of phosphorylated ubiquitin and of Lys63‐linked diubiquitin at the proximal, distal or both ubiquitins is reported. This enabled the examination of how such a modification alters E1‐E2 activities of the ubiquitination machinery. It is found that E1 charging was not affected, while the assembly of phosphorylated ubiquitin chains was differentially inhibited with E2 enzymes tested. Moreover, this study shows that phosphorylation interferes with the recognition of linkage specific antibodies and the activities of several deubiquitinases. Notably, phosphorylation in the proximal or distal ubiquitin unit has differential effects on specific deubiquitinases. These results support a unique role of phosphorylation in the dynamics of the ubiquitin signal.     

Chemistry and biology of the ubiquitin signal

Ubiquitination is one of the most utilized posttranslational modifications in eukaryotes and is involved in a wide range of cellular processes, but is mostly known as a signal for proteasomal degradation. Recently, it has become clear that the ubiquitin signal is far more complex and is dictated by the ubiquitin component and the substrate. The remarkable diversity of the ubiquitin signaling process has triggered an incredible amount of effort to investigate the role of ubiquitination on biological processes. However, despite more than three decades of studies, several important questions remain unanswered. A major hurdle is the inability to obtain homogeneous ubiquitin bioconjugates in sufficient amounts from cells or by application of the enzymatic machinery. Recent breakthroughs in chemical and semisynthetic strategies, however, offer solutions to these challenges. In this Review, we survey the fundamental biological aspects of the ubiquitin signal and present the emerging non-enzymatic approaches for overcoming these obstacles.     

Palladium in the Chemical Synthesis and Modification of Proteins

The field of site‐specific modification of proteins has drawn significant attention in recent years owing to its importance in various research areas such as the development of novel therapeutics and understanding the biochemical and cellular behaviors of proteins. The presence of a large number of reactive functional groups in the protein of interest and in the cellular environment renders modification at a specific site a highly challenging task. With the development of sophisticated chemical methodologies it is now possible to target a specific site of a protein with a desired modification, however, many challenges remain to be solved. In this context, transition metals in particular palladium‐mediated C−C bond‐forming and C−O bond‐cleavage reactions gained great interest owing to the unique catalytic properties of palladium. Palladium chemistry is being explored for protein modifications in vitro, on the cell surface, and within the cell. Very recently, palladium complexes have been applied for the rapid deprotection of several widely utilized cysteine protecting groups as well as in the removal of solubilizing tags to facilitate chemical protein synthesis. This Minireview highlights these advances and how the accumulated knowledge of palladium chemistry for small molecules is being impressively transferred to synthesis and modification of chemical proteins.     

Carnation‐like CuO Hierarchical Nanostructures Assembled by Porous Nanosheets for Nonenzymatic Glucose Sensing

Carnation‐like CuO hierarchical nanostructures assembled by ultrathin porous nanosheets were successfully fabricated via a facile solvothermal route followed with heat treatment. As‐prepared CuO nanostructures exhibited excellent catalytic activity toward glucose oxidation in the absence of any enzymes. Under the optimized conditions, the CuO‐based enzymeless glucose sensor showed high sensitivity of 3.15 mA mM^?1 cm^?2, low limit of detection (98 nM, S/N=3), good reproducibility, excellent selectivity and long‐time stability. The superb nonenzymatic glucose sensing performance of the CuO hierarchical nanostructures was attributed to the highly catalytically active sites at the edges and basal planes of the CuO nanosheets, facile transportation of analytes through the abundant mesopores and macropores, robust and stable hierarchical structure. Moreover, the CuO‐based enzymeless glucose sensor showed high accuracy and reliability in comparison with clinical glucometer for quantitative determination of glucose in human blood serum samples.     

A Water-Soluble Iridium Photocatalyst for Chemical Modification of Dehydroalanines in Peptides and Proteins

Dehydroalanine (Dha) residues are attractive noncanonical amino acids that occur naturally in ribosomally synthesised and post-translationally modified peptides (RiPPs). Dha residues are attractive targets for selective late-stage modification of these complex biomolecules. In this work, we show the selective photocatalytic modification of dehydroalanine residues in the antimicrobial peptide nisin and in the proteins small ubiquitin-like modifier (SUMO) and superfolder green fluorescent protein (sfGFP). For this purpose, a new water-soluble iridium(III) photoredox catalyst was used. The design and synthesis of this new photocatalyst, [Ir(dF(CF₃)ppy)₂(dNMe₃bpy)]Cl₃, is presented. In contrast to commonly used iridium photocatalysts, this complex is highly water soluble and allows peptides and proteins to be modified in water and aqueous solvents under physiologically relevant conditions, with short reaction times and with low reagent and catalyst loadings. This work suggests that photoredox catalysis using this newly designed catalyst is a promising strategy to modify dehydroalanine-containing natural products and thus could have great potential for novel bioconjugation strategies.     

Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins

In this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aqueous medium with excellent selectivity and quantitative conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biological systems. Additionally, fluorescent‐labeling studies demonstrated that the installation of a C‐terminal allenamide moiety onto various molecules of interest may supply a new methodology towards the site‐specific labeling of cysteine‐containing proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences.     

Chemical Modification of Proteins at Cysteine: Opportunities in Chemistry and Biology

Cys‐tematic modification : Cysteine is a versatile amino acid for selective chemical modification of proteins. Both chemical and biological innovations made possible by cysteine modification are highlighted in this Focus Review.

     

A New Pyridoxal Derivative for Transamination of N‐Terminus of Proteins

A new pyridoxal‐5‐phosphate (PLP) derivative FHMDP was developed for the transamination of different peptides with three most hindered amino acid residues (Leu, Ile, Val) as their N‐terminus. Compared to the previously reported reactions of PLP derivatives, the N‐terminus transamination could be accomplished efficiently with the new compound.     

Single-Step N-Terminal Modification of Proteins via a Bio-Inspired Copper(II)-Mediated Aldol Reaction

The chemical modification of proteins is an effective technique for manipulating the properties and functions of proteins, and for creating protein-based materials. The N-terminus is a promising target for single-site modification that provides modified proteins with uniform structures and properties. In this paper, a copper(II)-mediated aldol reaction with 2-pyridinecarboxaldehyde (2-PC) derivatives is proposed as an operationally simple method to selectively modify the N-terminus of peptides and proteins at room temperature and physiological pH. The copper(II) ion activates the N-terminal amino acids by complexation with an imine of the N-terminal amino acid and 2-PCs, realizing the selective formation of the nucleophilic intermediate at the N-terminus. This results in a stable carbon-carbon bond between the 2-PCs and the α-carbon of various N-terminal amino acids. The reaction is applied to four different proteins, including biopharmaceuticals such as filgrastim and trastuzumab. The modified trastuzumab retains the human epidermal growth factor receptor 2 recognition activity.